The present invention relates to apparatus for counting and classifying particles by directing a laser beam toward the particles and sensing the radiation by means of a detector.
For various purposes, it is necessary to count and record the size distribution of cells and particles with simultaneous classification according to certain cell characteristics. However, numerous problems have been encountered in procedures thus far devised for performing these operations.
One known process based on the Coulter principle provides for electronic measurement of the cell volume on the basis of changes in resistance of an electrolyte liquid during passage of the cells through an opening in a partition.
It has also been proposed to employ optical flow-through methods which entail fluorescence measurements with colored particles and differentiation according to the intensity of the fluorescence, or scattered light measurements in which a coherent light source is scattered at particles, or measurements of the absorption of the entire cell on object carriers. In all these optical processes the measuring volume is greater, however, than the cell or particle to be measured, respectively.
In measurement procedures according to the Coulter principle, the measured value depends on the geometry of the measuring opening in the partition and on the location of the path of travel of the particles through the measuring opening. No information other than cell volume can be obtained about the particles. Moreover, there exists a danger of the measuring opening becoming clogged and the maximum cell diameter is limited to 50% of the measuring opening. The result is a low counting rate which is still dependent on particle size.
Fluorescence measurements have the drawback that the measured value is dependent on the coloration processes, i.e., different measuring series cannot be directly compared with one another and fluorescence colorations of special cell characteristics can often not be produced at all. In the case of scattered light measurements it is necessary, in order to record a size distribution, to simultaneously effect measurements at various spatial angles. This has the result that only size distributions up to a maximum of about 10.mu. can be derived from scatter data. In the practice of both of these measuring methods, the particles are present in suspension and the optical quality of the suspension stream is also not optimally adapted to the index of refraction of the cells.
Absorption measurements have previously been successful only when the measuring field is larger than the cell cross section. The cells are here applied to object carriers, and this results in low counting and analysis speeds since the object carrier must, inter alia, be moved mechanically.